新型PLA/BMG多孔复合生物活性材料的制备及相关研究
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摘要
第一章PLMBMG多孔复合生物活性材料的制备及理化性质检测
目的：制备骨基质明胶(BMG)，采用超临界二氧化碳法与聚乳酸(PLA)复合制备PLA／BMG多孔复合生物活性骨植入材料，通过理化性能检测选择PLA／BMG最佳复合比例，为后续实验研究提供依据。
方法：选取合格供体皮质骨，按山西省医用组织库同种骨生产标准进行冷冻、清洗，粉碎后选择粒径大小为50μm～200μm的骨粉，将其进行脱脂、脱钙等处理制备成骨基质明胶(BMG)；将PLA与BMG按体积比10：0、9：1、8：2，7：3，6：4进行混匀，再加入粒径为100μm～200μm的NaCl颗粒作为造孔剂(与复合材料的质量比为3：1)，置于超临界二氧化碳反应装置内，将其密封后用液泵将冷凝到0℃以下的CO_2压进反应釜内，当反应釜内压力达到20MPa时，开始升温，使反应釜内温度达到36℃，调节釜内的压力和温度，保持温度变化不超过士0.5℃，压力变化为±0.1MPa。经过30 min后，以5 MPa／min的速率放气，打开反应釜，取出样品，去离子水浸沥NaCl 48h，冷冻干燥，包装后进行辐照灭菌备用。通过大体观察、孔隙率测定，力学检测、SEM观察评价其理化性能，并结合体外细胞相容性检测选择最佳复合比例。
结果：所制备PLA／BMG复合材料大体观察结构疏松，有较好的孔隙率，类似人体松质骨样结构；不同比例复合材料孔隙率均大于60％，且随着BMG含量的增加而增大；PLA／BMG复合材料的抗压强度及弹性模量均随BMG含量的增加而逐渐降低，当BMG含量大于30％时，力学性能下降显著；细胞相容性方面，聚乳酸材料明显的抑制了大鼠L6成肌细胞的增殖，但随着BMG比例的增大，细胞相容性逐渐转好，达到40％时，基本与正常培养基组相同，对细胞增殖无明显影响。扫描电镜观察所制备含30％BMG的复合材料BMG与PLA混合均匀，BMG与PLA结合较为致密，材料孔隙分布均匀，孔隙大小大约在200μm-400μm之间，孔洞连通性好，且在孔隙内壁上，仍可见大量孔径为10μm左右的小孔隙。
结论：采用SC-CO_2法制备的PLA／BMG多孔复合支架材料中BMG的比例与材料的孔隙率及细胞相容性成正相关，与力学性能成负相关；含30％BMG的复合材料具有良好的综合性能，为SC-CO_2法制备PLA／BMG的最佳复合比例。
第二章PLA／BMG多孔复合材料生物相容性检测
目的：采用动物体内实验和细胞体外实验评价所制备PLA／BMG多孔复合生物活性材料的组织生相容及细胞相容性，为PLA／BMG复合材料提供生物相容性实验数据。
方法：体外细胞毒性评价：参照中华人民共和国医疗器械生物学评价标准，将所制备的PLA／BMG复合材料、PLA材料按质量与浸提液0.1g／ml的比例，浸入不含血清的DMEM高糖培养基的封闭容器内，37℃培养箱72h，取出材料，将浸提液离心，取上清液并加入10％的胎牛血清；将对数生长期的小鼠MC3T3-E1成骨前体细胞制备为1.5×10~5／ml细胞悬液，并接种于96孔培养板，单纯DMEM高糖培养液组作阴性对照，不含细胞孔作为空白对照，每孔200μl，培养24h待细胞贴壁后，按组别分别加入200μl材料浸提液，置37℃5％CO_2培养箱中继续培养，每2d更换浸提液一次。分别于培养1、2、3、4、5、6、7d后，每孔加入30μl 2mg／ml的MTT溶液，继续培养4h，吸出孔内培养液后，每孔加入150μl DMSO，振荡使结晶物溶解，酶标仪检测各孔OD值，测试波长为570 nm，计算细胞增殖率并转换为细胞毒性级。PLA／BMG复合材料细胞亲和性测定：将PLA／BMG修剪成3mm×3mm×3mm大小，放置于6孔培养板；传代至第3代的MC3T3-E1细胞以1×10~4／ml接种于已放置材料的培养板，加DMEM高糖培养基至完全淹没材料，于接种后1、3、5天倒置相差显微镜观察细胞生长及与材料贴附情况，并于第5天取出材料，扫描电镜观察细胞贴附情况。生物相容性体内评价：Wistar大鼠20只，无菌条件下，分别将PLA及PLA／BMG植于脊柱左、右侧的皮下，及左右后肢的股肌袋内，术后行大体观察术后饮食、活动、切口反应等，于术后2、4、6、8周随机处死5只动物，切取包括植入材料及周围组织的复合组织块，常规组织切片，HE染色，光镜下观察材料及周围组织的组织学变化。
结果：体外细胞毒性检测：PLA／BMG复合材料浸提液培养细胞1～7天内细胞增殖率均大于100％，细胞毒性级均为0级；而PLA材料组第2、4、7天细胞增殖率小于100％，细胞毒性级为1级。材料与细胞复合培养第1天，材料周围基本无细胞贴附，至第3天，材料侧缘可见少量细胞贴附，材料周围细胞形态正常，第5天，材料侧缘贴附细胞增多，周围细胞生长状态良好，部分部位呈汇聚状态；扫描电镜观察可见，材料表面及材料孔隙内壁均有细胞贴附，细胞呈三角形或多边形，表面有较多的突起。有些部位多个细胞在材料表面汇合成片状分布，细胞与材料贴附紧密，细胞表面有泡状突起，且可见分裂相细胞。皮下及肌内植入实验显示PLA组材料被纤维结缔组织所包裹，周围组织及材料内部结缔组织内含有较多的淋巴细胞、中性粒细胞等炎症细胞，结缔组织向材料内部生长缓慢；PLA／BMG组纤维组织包裹不明显，结缔组织易于向材料中心长入，材料周围组织及材料内部长入的结缔组织内炎症细胞少见，可观察到间充质细胞向材料中散在的BMG贴附。
结论：SC-CO_2法制备的PLA／BMG复合材料对MC3T3-E1细胞有较好的细胞亲和性，细胞毒性级别为0级，具有良好的细胞相容性；皮下及肌内植入均显示PLA／BMG复合材料较单纯PLA材料具有更好的组织相容性。
第三章PLA／BMG多孔复合材料生物活性检测
目的：体外将PLA／BMO复合材料与小鼠MC3T3-E1成骨前体细胞共培养，通过对钙结节、钙含量与ALP活性的测定，间接评价PLA／BMG多孔复合材料的骨诱导活性，为进一步实验提供实验依据。
方法：实验分组：DMEM组为培养基对照组(不含任何材料)；PLA／BMG组为每孔加入100μg粉碎的PLA／BMG复合材料；PLA组为每孔加入100μg粉碎的PLA；每组设6复孔。将小鼠MC3T3-E1成骨前体细胞适当传代后，稀释制成2×10~6／ml细胞悬浮液，每孔20μl接种于含不同组别材料的24孔培养板内，细胞粘壁后，分别加入1ml含10μmol／mlβ-甘油磷酸及5μg／ml抗坏血酸的DMEM高糖培养基。每3d换液一次，每次换液量为培养液的2／3，培养2周后，消化收集细胞。钙结节测定：PBS沖洗收集细胞后的培养板，福尔马林溶液固定后，1％茜素红溶液染色，倒置相差显微镜观察，数码相机拍摄每个培养孔图像，图像处理与分析软件测定被染色面积占培养孔总面积的百分比，即为钙化结节面积百分比。钙含量及碱性磷酸酶(ALP)活性测定：0.2％的NP-40重新悬浮收集细胞，在冰浴中超声裂解，将细胞裂解液严格按照钙含量及ALP活性测定试剂盒说明书进行钙含量及ALP活性的检测。应用方差分析和Tamhane's T2检验进行统计学分析。
结果：PLMBMG组ALP活性、钙含量及钙化面积均值分别为[(325.594±70.40)U／g prot、(3.51±1.64)mmol／g prot)、(42.98±4.44)％]均高于PLA组[(63.624±30.01)U／gprot、(1.04±0.21)mmol／g prot、(9.55±1.94)％](P＜0.05)及DMEM培养基组[(2.40±1.47)U／g prot、(0.70+0.24)mmol／g prot、(0.86±0.41)％](P＜0.05)，同时PLA组的ALP活性及钙化结节面积与DMEM培养基组也存在显著差异(P＜0.05)。
结论：采用SC-CO_2法制备的PLMBMG多孔复合支架材料具有良好的骨诱导活性，好于单纯PLA材料，有可能作为一种有前景的骨植入材料及骨组织工程支架材料。
第四章PLA／BMG多孔复合材料修复兔桡骨节段性骨缺损的实验研究
目的：建立兔桡骨节段性骨缺损模型并将PLA／BMG多孔复合材料植入，评价其修复节段性骨缺损的能力，为可能的临床应用提供实验依据。
方法：选用新西兰大白兔24只，腹腔麻醉后，无菌操作暴露桡骨中段，牙科钻截取兔双侧桡骨中段，造成约12mm骨缺损，按组别分别植入各组材料，于术后4w、8w、12w分别取材，每次8只。实验分组：空白对照组，骨缺损处不植入任何材料作为阴性对照；PLA材料组，骨缺损处植入PLA材料；PLA／BMG复合材料组，骨缺损处植入PLA／BMG多孔复合材料；自体骨组，骨缺损处将截取的自体骨再植入骨缺损处，作为阳性对照。X光检查：动物于取材时间，行桡骨正位X线片检查，观察不同时间点骨缺损愈合情况。根据Lane等的X线片评分标准对各组移植区骨形成、骨连接和骨塑形情况进行评分，并进行统计学检验。组织学观察：截取桡骨植骨部位，有机树脂包埋，用硬组织切片机制作厚度为5μm切片标本，进行HE染色，光镜下观察骨形成及材料降解情况。
结果：大体观察：除空白对照组1例，PLA组2例出现骨折外，其余动物术后状态良好，术肢无明显并发症发生。X线观察：空白对照组术后4w骨缺损两侧骨断端锐利，没有任何骨痂形成，骨缺损区明显可见；术后8w，骨缺损两端可见少量低密度骨痂形成，骨缺损区缩小；术后12w，缺损区仍空虚，两骨断端细长骨痂形成，骨缺损区进一步缩小，未见连续性骨痂，骨髓腔封闭，为骨不连。PLA组术后4w骨缺损两端有少量低密度的骨痂形成，缺损区呈现密度低于骨干的雾状影；术后8w，两断端在近尺骨侧出现高密度骨痂连接，远离尺骨侧骨缺损仍清晰可见；术后12w，骨缺损两端基本连接，骨密度不均，周围有骨痂形成，骨髓腔没有再通。PLA／BMG组术4周两断端有骨痂包裹，缺损区呈现大片云雾状高密度影，骨缺损区不易辨认；术后8w，骨缺损两端均基本连接，缺损区内呈现高密度影；术后12w，新骨桥接骨缺损，密度较均一，皮质骨连续，形成连接性骨质，骨缺损基本完全愈合，髓腔再通。自体骨组术后4w骨缺损区自体骨仍清晰可见，截骨端有少量骨痂形成；术后8w，植入自体骨段与宿主骨两端连接，连接处呈现高密度影，有较多的骨痂形成；术后12w，新骨桥接骨缺损，密度较8周时增高，但不均匀，周围有大量骨痂形成，髓腔再通。X线评分：术后4w、8w、12w，PLA／BMG复合材料组(4.25、7.25、9.50)与自体骨组(4.50、8.00、10.25)在修复骨缺损上无显著性差异，而PLA／BMG组和自体骨组修复骨缺损效果优于空白对照组(0.25、0.50、1.25)和PLA组(1.00、3.50、4.25)，存在显著性差异(P＜0.05)；PLA组仅在第8w显示出修复效果好于空白对照组(P＜0.05)，而4w与12w两组间无显著性差异。组织学观察：空白对照组骨缺损被纤维组织填充，无新骨组织形成。PLA组材料逐渐被纤维组织包裹吸收，成骨不活跃；PLA／BMG组材料可见大量新生骨向材料内部长入，间充质细胞向BMG贴附，材料与宿主骨结合紧密；自体骨组材料结合部有大量骨痂形成，骨组织已进行改建与重塑。
结论：PLA／BMG多孔复合材料与自体骨具有相同的修复节段性骨缺损能力，优于PLA材料的修复能力。
Part 1 Preparation of the Porous PLA/BMG Composite Biomaterials and its Physical and Chemical Characteristics Study
Objective: To develop a novel porous poly (lactic-acid) (PLA)/bone matrix gelatin (BMG) bioactive composite biomaterial with supercritical carbon dioxide fluid technique (SC-CO_2) and to choose the optimal PLA/BMG proportion by evaluating its physical and chemical characteristics and cellular compatibility.
Methods: The bone allografts procured from the qualified donor were processed into bone powder with the size of 50μm～200μm by deep-freezing, cleaning according to the processing standards of bone allograft in Shanxi Provincial Tissue Bank. Then the powder was processed into Bone Matrix Gelatin (BMG) by defatting and demineralization as described by Urist. The PLA and BMG, mixed with the PLA:BMG proportion of 9: 1、8: 2, 7: 3, 6: 4, and the 100μm～200μm NaCl was added in the SC-CO_2 equipment in order to make pores. Tightly closing the reactor kettle and CO_2 under 0℃was pumped into kettle. When the internal pressure reached 20MPa, the temperature was regulated at 36℃. The pressure was kept in (20±0.1) MPa and the temperature in (36±0.5)℃. After 30 minutes, the gas was released at the rate of 5 MPa/min.Opening the reacting kettle and the samples were taken out. After the samples were leached in deionised water to remove the NaCl in material, they were packaged and sterilized by radiation. The macroscopic observation, porosity, mechanics and scanning electron micrograph (SEM) were done to evaluate the physical and chemical properties. Then referring to the cellular compatibility, the optimal proportion was chosen.
Results: In macroscopic observation, the PLA/BMG composite biomaterial, prepared with the SC-CO_2 technique, have porous structure and good interconnective pores. The structure is like that of human cancellous bone. The porosity was more than 60 % and increasing with the BMG proportion being improved. While the compressive strength and elastic modulus was in inverse proportion to the proportion of BMG in PLA/BMG. When the BMG proportion was more than 30 %, the mechanics properties dropped significantly. In cell compatibility, PLA biomaterial did harm to proliferation of rat L6 cells. With the increasement of BMG proportion, cell compatibility was getting better. When the proportion reached 40 %, there was no difference between the PLA/BMG composite biomaterial group and the culture medium group. The SEM of PLA/BMG with 30 % BMG showed PLA and BMG were mixed uniformly, the pores diametesr were between 200μm and 400μm and the pores were distributed uniformly and interconnected each other. On the wall of pores, there still a number of about 10μm pores.
Conclusion: The percent of BMG in the BLA/BMG composite biomaterials prepared with SC-CO_2 technique was in direct proportion to its cellular compatibility and porosity, and in inverse proportion to its mechanics. The results show the PLA/BMG containing 30 % BMG was the optimal.
Part 2 Evaluation of Biocompatibility on the Porous PLA/BMG Composite Biomaterials
Objective: To evaluate the tissue biocompatibility in vivo by animal experiments and cell biocompatibility in vitro by cell experiments and provide the biocompatibility datas for PLA/BMG composite biomaterial.
Methods: Tests for cytotoxicity in vitro methods: Referring to the biological evaluation of medical devices-Tests for cytotoxicity: in vitro methods, the PLA/BMG and PLA porous biomaterials were immersed in DMEM culture medium with 0.1 g biomaterial each ml DMEM. After 72 hours in 37℃, the supernatants were collected by centrifuge. 200μl of the MC3T3-E1 cell suspension with a cell density of 5×10~5 cells/disk was seeded evenly into each well in 96-Well Plate. When the cell adhered after 24 hours, the culture medium in wells was removed and 200μl supernatant were added according to groups respectively, with DMEM culture medium being the control group and no cells well being the blank control group. The cells were cultured in 37℃humidified atmosphere of 5% CO_2. The cell culture mediums were exchanged every two days. On the 1~(st) day, 2~(nd) day, 3~(rd) day, 4~(th) day, 5~(th) day, 6~(th) day and 7~(th) day respectively, 30μl 2mg/ml MTT solution was added into each well. After being incubated for 4 hours, 150μl DMSO was added and the plate was shaken for 10 minutes. The OD of the medium was read at 570 nm with a plate reader. The cell proliferation rates were calculated and transformed into the toxicity grades. The cellular affinity of PLA/BMG composite biomaterial: The 3 mm×3 mm×3 mm PLA/BMG blocks processed were placed in 96-Well Plate. 500μl of 1×10~4/ml MC3T3-E1 cell suspension was seeded into each well and DMEM was added to immerse the PLA/BMG blocks. Cell morphology and adhering to materials was observed on the 1~(st), 3~(rd), 5~(th) day after seeding under phase contrast microscope and on the 5~(th) day under SEM. Biological evaluation in vivo: 20 Wistar mice, PLA biomaterials were implanted both in nape subcutaneous tissue and thigh muscle on the left side, and PLA/BMG biomaterials on the right side. The gross observation about animal situations was made. At the 2~(nd), 4~(th), 6~(th), 8~(th) week after implantation, the 5 mice were chosen to be killed randomly. The implanted material and surrounding tissue were procured and stained with HE. The histological changes were made observation under light microscope.
Results: Tests for cytotoxicity in vitro methods: In PLA/BMG group, the cell proliferation rates were over 100 % and the cell cytotoxic grades were Grade 0 from the 1~(st) day to the 7~(th) day. While in PLA group, the cell proliferation rates were less than 100 % and the cell cytotoxic grades were Grade I on the 2~(nd), 4~(th) and 7~(th) day. The cellular affinity of PLA/BMG composite biomaterial: on the 1~(st) day after seeding, there were few cells on PLA/BMG. On the 3~(rd) day, a few cells adhered to the edge of PLA/BMG and the cells surrounding kept normal morphology. On the 5~(th) day, the number of cell adhered to PLA/BMG increased, even converged somewhere. SEM showed there were cells both on the surface and in the pores of PLA/BMG. The cellular morphology was triangle or polygon with abundant microvillus on the surface. Some cells connected and overlapped by protrusions. The cells adhered to the surface of PLA/BMG tightly. Some cells were keeping at mitosis phase. Biological evaluation in vivo showed PLA were enclosed with connective tissue containing a lot of lymphocytes and neutrophilic granulocytes. The cells and tissue grew into PLA slowly. The PLA/BMG materials were enclosed with little connective tissue including a few inflammatory cells. The surrounding tissue immersed into the center of PLA/BMG easily. Some mesenchymal cells migrated to the edge of BMG.
Conclusion: the PLA/BMG composite biomaterials prepared with SC-CO_2 technique is of nice cellular affinity. The cytotoxicity grade is Grade 0, better than that of PLA. It shows good cell compatibility. Biological evaluation in vivo also suggests the tissue compatibility of PLA/BMG is better than that of PLA.
Part 3 Experimental Study on Osteoinductive Activity of the Porous PLA/BMG Composite Biomaterials
Objective: To evaluate the osteoinductive activity of PLA/BMG by calcium depositions, ALP activity and Ca content by Mouse osteoblast-like MC3T3-E1 cells be cultured in DMEM with the different biomaterials.
Methods: The PLA/BMG group: with 100μg of the scrushed PLA/BMG each well; The PLA group: with 100μg of the scrushed PLA each well. The DMEM group serves the control. There were 6 wells in each group. Cells suspensions were prepared and adjusted to 2×10~6 cells/ml. 20μl cells suspension was delivered into each well of 24 well plate with the different group materials. After the cells were adhered, 1 ml of culture medium containing 10μmol/ml disodiumβ-glycerophosphate (β-GP) and 5μg/ml vitamin C was added. 2/3 of Culture medium was changed every 3 days. After 2 weeks, the cells were collected. Calcification depositions assay: After being washed three times with PBS buffer, the cell cultures were fixed with formalin and stained by alizarin red solution. The microscopic images were taken with the converse phase microscope and the macroscopic ones with the digital camera. The percentage of the stained area, standing for the calcification deposition, was measured with the image processing and analysis software. ALP activity and Calcium content measurement: After the harvested cells were washed twice with Hanks solution. The cells were resuspended in 1 ml of 0.2 % Nonidet P-40 (NP-40) and sonicated in an ice bath for 2 min using an ultrasonic processor. The measurement of the ALP activity and calcium content were taken according to the instructions of kits.
Results: The ALP activity, Ca content and percent of calcification area in PLA/BMG group [ (325.59±70.40) U/g prot、(3.51±1.64) mmol/g prot)、(42.98±4.44) % ] were more than those in PLA group [ (63.62±30.01) U/gprot、(1.04±0.21) mmol/g prot, (9.55±1.94)% ] and DMEM control group [ (2.40±1.47) U/g prot、(0.70±0.24) mmol/g prot、(0.86±0.41) % ] (P＜0.05). Meanwhile, there was statistic difference between PLA group and DMEM group in the ALP activity and calcification area.
Conclusion: The PLA/BMG prepared by SC-CO_2 technique has good osteoinductive activity, better than that of PLA. And it is worth studying further as bone biomaterial and bone tissue engineering scaffold.
Part 4 Experimental Study of the Porous PLA/BMG Composite Biomaterials
for the Repair Radial Segmental Bone Defect of Rabbit
Objective: To evaluate the repair of rabbit radial defects by PLA/BMG biomaterials prepared by SC-CO_2 technique and provide the experimental date for the possible clinical applications.
Methods: Twenty four New Zealand male rabbits with the Wt of about 2.5 kg were obtained for this study. After the rabbits were anesthetized and the forelegs were sterilized, the diaphysis of both radii were exposed through a longitudinal extensile incision, the periosteum was elevated circumferentially, and the 12 mm long middle shaft of the radius was cut with a power-driven oscillating saw. Then a 12 mm bone defect was created. The biomaterials were filled according to the different groups. Then the wound was closed in layers and the animals were put back into their cages. The animals were divided into 2 groups randomly. In one group, nothing was filled in defect on the left and the bone autograft cut was re-implanted on the right; in another group, PLA/BMG was filled on the left and PLA/BMG was done on the right. 4 animals of each group were put to death at week 4, 8, 12 after operation. Radiographic observation and evaluation: Radiographs were taken when the animals were put to death at week 4, 8, 12 respectively. The situation of bone reconstruction were observed each group and evaluated in a blind condition according to the Standard of Lane's X-ray Scores. Histological observation: the radial samples from rabbits were harvested and fixed in formalin solution. Then the dehydrated samples were embedded with plastic and sectioned longitudinally by 5 mm thickness, stained with hematoxylin and eosin, and finally observed under light microscope for the formation of the new bone and the degradation of the material.
Results: Gross observation: except forl bone fracture in the blank control group and 2 fracture in the PLA group, all the animal were in good condition after operation and the operated limbs was uneventful with no detectable complications. Radiographic findings: In the blank control group, at 4 week postoperatively, little callus formed and the bone defect was clear; at 8 week postoperatively, a little callus with low-density was seen and the length of bone defect became shorter; at 12 week postoperatively, some slim callus formed at the both ends of broken bone and the defect was seen easily. In the PLA group, at 4 week postoperatively, a little callus with low-density formed at the ends and some fog-like image was seen in the defect; At 8 week postoperatively, the callus at the both ends met near to the ulna, but the defect was clear far to the ulna; at 12 week postoperatively, the both ends were connected with uneven shadow, little callus formed circumferentially and the cavitas medullaris did not recanalize. In the PLA/BMG group, at 4 week postoperatively, the both ends were enclosed with callus and the defect full of high-density shadow can not be identified easily; at 8 week postoperatively, the both ends met by high-density shadow; at 12 week postoperatively, with the new bone with uniform density bridging the both ends, the bone was reconstructed and the cavitas medullaris recanalized. In the autograft group, at 4 week postoperatively, the implanted bone could be identified and some callus formed at the broken ends; at 8 weeks postoperatively, the implanted bone were connected with the host bone by high-density callus; at 12 week postoperatively, the both ends were bridged with uneven high-density callus and the avitas medullaris recanalized. Radiographic evaluation: The PLA/BMG group and bone autograft group had no statistic difference at week 4, 8, 12 postoperatively. Which was better than both the PLA group and the blank control group (P＜0.05). There was significant difference at week 8 (P＜0.05) and no difference at week 4 and 8 between the PLA group and the blank control group. Histological Findings: In the blank group, the defect was filled with fiber connective tissue and no new bone formed. In the PLA group, the PLA were enveloped with connective tissue with little new bone formation. In the PLA/BMG group, the PLA/BMG incorporated with the host bone tightly. The new bone immersed into the PLA/BMG and some mesenchymal cells attached the BMG. In bone autograft group, large amounts of callus formed at the interface and the bone had reconstructed and remodeled.
Conclusions: The ability to repair bone defect of the PLA/BMG is same to that of bone autograft and better than that of the PLA.
目的：制备骨基质明胶(BMG)，采用超临界二氧化碳法与聚乳酸(PLA)复合制备PLA／BMG多孔复合生物活性骨植入材料，通过理化性能检测选择PLA／BMG最佳复合比例，为后续实验研究提供依据。
方法：选取合格供体皮质骨，按山西省医用组织库同种骨生产标准进行冷冻、清洗，粉碎后选择粒径大小为50μm～200μm的骨粉，将其进行脱脂、脱钙等处理制备成骨基质明胶(BMG)；将PLA与BMG按体积比10：0、9：1、8：2，7：3，6：4进行混匀，再加入粒径为100μm～200μm的NaCl颗粒作为造孔剂(与复合材料的质量比为3：1)，置于超临界二氧化碳反应装置内，将其密封后用液泵将冷凝到0℃以下的CO_2压进反应釜内，当反应釜内压力达到20MPa时，开始升温，使反应釜内温度达到36℃，调节釜内的压力和温度，保持温度变化不超过士0.5℃，压力变化为±0.1MPa。经过30 min后，以5 MPa／min的速率放气，打开反应釜，取出样品，去离子水浸沥NaCl 48h，冷冻干燥，包装后进行辐照灭菌备用。通过大体观察、孔隙率测定，力学检测、SEM观察评价其理化性能，并结合体外细胞相容性检测选择最佳复合比例。
结果：所制备PLA／BMG复合材料大体观察结构疏松，有较好的孔隙率，类似人体松质骨样结构；不同比例复合材料孔隙率均大于60％，且随着BMG含量的增加而增大；PLA／BMG复合材料的抗压强度及弹性模量均随BMG含量的增加而逐渐降低，当BMG含量大于30％时，力学性能下降显著；细胞相容性方面，聚乳酸材料明显的抑制了大鼠L6成肌细胞的增殖，但随着BMG比例的增大，细胞相容性逐渐转好，达到40％时，基本与正常培养基组相同，对细胞增殖无明显影响。扫描电镜观察所制备含30％BMG的复合材料BMG与PLA混合均匀，BMG与PLA结合较为致密，材料孔隙分布均匀，孔隙大小大约在200μm-400μm之间，孔洞连通性好，且在孔隙内壁上，仍可见大量孔径为10μm左右的小孔隙。
结论：采用SC-CO_2法制备的PLA／BMG多孔复合支架材料中BMG的比例与材料的孔隙率及细胞相容性成正相关，与力学性能成负相关；含30％BMG的复合材料具有良好的综合性能，为SC-CO_2法制备PLA／BMG的最佳复合比例。
第二章PLA／BMG多孔复合材料生物相容性检测
目的：采用动物体内实验和细胞体外实验评价所制备PLA／BMG多孔复合生物活性材料的组织生相容及细胞相容性，为PLA／BMG复合材料提供生物相容性实验数据。
方法：体外细胞毒性评价：参照中华人民共和国医疗器械生物学评价标准，将所制备的PLA／BMG复合材料、PLA材料按质量与浸提液0.1g／ml的比例，浸入不含血清的DMEM高糖培养基的封闭容器内，37℃培养箱72h，取出材料，将浸提液离心，取上清液并加入10％的胎牛血清；将对数生长期的小鼠MC3T3-E1成骨前体细胞制备为1.5×10~5／ml细胞悬液，并接种于96孔培养板，单纯DMEM高糖培养液组作阴性对照，不含细胞孔作为空白对照，每孔200μl，培养24h待细胞贴壁后，按组别分别加入200μl材料浸提液，置37℃5％CO_2培养箱中继续培养，每2d更换浸提液一次。分别于培养1、2、3、4、5、6、7d后，每孔加入30μl 2mg／ml的MTT溶液，继续培养4h，吸出孔内培养液后，每孔加入150μl DMSO，振荡使结晶物溶解，酶标仪检测各孔OD值，测试波长为570 nm，计算细胞增殖率并转换为细胞毒性级。PLA／BMG复合材料细胞亲和性测定：将PLA／BMG修剪成3mm×3mm×3mm大小，放置于6孔培养板；传代至第3代的MC3T3-E1细胞以1×10~4／ml接种于已放置材料的培养板，加DMEM高糖培养基至完全淹没材料，于接种后1、3、5天倒置相差显微镜观察细胞生长及与材料贴附情况，并于第5天取出材料，扫描电镜观察细胞贴附情况。生物相容性体内评价：Wistar大鼠20只，无菌条件下，分别将PLA及PLA／BMG植于脊柱左、右侧的皮下，及左右后肢的股肌袋内，术后行大体观察术后饮食、活动、切口反应等，于术后2、4、6、8周随机处死5只动物，切取包括植入材料及周围组织的复合组织块，常规组织切片，HE染色，光镜下观察材料及周围组织的组织学变化。
结果：体外细胞毒性检测：PLA／BMG复合材料浸提液培养细胞1～7天内细胞增殖率均大于100％，细胞毒性级均为0级；而PLA材料组第2、4、7天细胞增殖率小于100％，细胞毒性级为1级。材料与细胞复合培养第1天，材料周围基本无细胞贴附，至第3天，材料侧缘可见少量细胞贴附，材料周围细胞形态正常，第5天，材料侧缘贴附细胞增多，周围细胞生长状态良好，部分部位呈汇聚状态；扫描电镜观察可见，材料表面及材料孔隙内壁均有细胞贴附，细胞呈三角形或多边形，表面有较多的突起。有些部位多个细胞在材料表面汇合成片状分布，细胞与材料贴附紧密，细胞表面有泡状突起，且可见分裂相细胞。皮下及肌内植入实验显示PLA组材料被纤维结缔组织所包裹，周围组织及材料内部结缔组织内含有较多的淋巴细胞、中性粒细胞等炎症细胞，结缔组织向材料内部生长缓慢；PLA／BMG组纤维组织包裹不明显，结缔组织易于向材料中心长入，材料周围组织及材料内部长入的结缔组织内炎症细胞少见，可观察到间充质细胞向材料中散在的BMG贴附。
结论：SC-CO_2法制备的PLA／BMG复合材料对MC3T3-E1细胞有较好的细胞亲和性，细胞毒性级别为0级，具有良好的细胞相容性；皮下及肌内植入均显示PLA／BMG复合材料较单纯PLA材料具有更好的组织相容性。
第三章PLA／BMG多孔复合材料生物活性检测
目的：体外将PLA／BMO复合材料与小鼠MC3T3-E1成骨前体细胞共培养，通过对钙结节、钙含量与ALP活性的测定，间接评价PLA／BMG多孔复合材料的骨诱导活性，为进一步实验提供实验依据。
方法：实验分组：DMEM组为培养基对照组(不含任何材料)；PLA／BMG组为每孔加入100μg粉碎的PLA／BMG复合材料；PLA组为每孔加入100μg粉碎的PLA；每组设6复孔。将小鼠MC3T3-E1成骨前体细胞适当传代后，稀释制成2×10~6／ml细胞悬浮液，每孔20μl接种于含不同组别材料的24孔培养板内，细胞粘壁后，分别加入1ml含10μmol／mlβ-甘油磷酸及5μg／ml抗坏血酸的DMEM高糖培养基。每3d换液一次，每次换液量为培养液的2／3，培养2周后，消化收集细胞。钙结节测定：PBS沖洗收集细胞后的培养板，福尔马林溶液固定后，1％茜素红溶液染色，倒置相差显微镜观察，数码相机拍摄每个培养孔图像，图像处理与分析软件测定被染色面积占培养孔总面积的百分比，即为钙化结节面积百分比。钙含量及碱性磷酸酶(ALP)活性测定：0.2％的NP-40重新悬浮收集细胞，在冰浴中超声裂解，将细胞裂解液严格按照钙含量及ALP活性测定试剂盒说明书进行钙含量及ALP活性的检测。应用方差分析和Tamhane's T2检验进行统计学分析。
结果：PLMBMG组ALP活性、钙含量及钙化面积均值分别为[(325.594±70.40)U／g prot、(3.51±1.64)mmol／g prot)、(42.98±4.44)％]均高于PLA组[(63.624±30.01)U／gprot、(1.04±0.21)mmol／g prot、(9.55±1.94)％](P＜0.05)及DMEM培养基组[(2.40±1.47)U／g prot、(0.70+0.24)mmol／g prot、(0.86±0.41)％](P＜0.05)，同时PLA组的ALP活性及钙化结节面积与DMEM培养基组也存在显著差异(P＜0.05)。
结论：采用SC-CO_2法制备的PLMBMG多孔复合支架材料具有良好的骨诱导活性，好于单纯PLA材料，有可能作为一种有前景的骨植入材料及骨组织工程支架材料。
第四章PLA／BMG多孔复合材料修复兔桡骨节段性骨缺损的实验研究
目的：建立兔桡骨节段性骨缺损模型并将PLA／BMG多孔复合材料植入，评价其修复节段性骨缺损的能力，为可能的临床应用提供实验依据。
方法：选用新西兰大白兔24只，腹腔麻醉后，无菌操作暴露桡骨中段，牙科钻截取兔双侧桡骨中段，造成约12mm骨缺损，按组别分别植入各组材料，于术后4w、8w、12w分别取材，每次8只。实验分组：空白对照组，骨缺损处不植入任何材料作为阴性对照；PLA材料组，骨缺损处植入PLA材料；PLA／BMG复合材料组，骨缺损处植入PLA／BMG多孔复合材料；自体骨组，骨缺损处将截取的自体骨再植入骨缺损处，作为阳性对照。X光检查：动物于取材时间，行桡骨正位X线片检查，观察不同时间点骨缺损愈合情况。根据Lane等的X线片评分标准对各组移植区骨形成、骨连接和骨塑形情况进行评分，并进行统计学检验。组织学观察：截取桡骨植骨部位，有机树脂包埋，用硬组织切片机制作厚度为5μm切片标本，进行HE染色，光镜下观察骨形成及材料降解情况。
结果：大体观察：除空白对照组1例，PLA组2例出现骨折外，其余动物术后状态良好，术肢无明显并发症发生。X线观察：空白对照组术后4w骨缺损两侧骨断端锐利，没有任何骨痂形成，骨缺损区明显可见；术后8w，骨缺损两端可见少量低密度骨痂形成，骨缺损区缩小；术后12w，缺损区仍空虚，两骨断端细长骨痂形成，骨缺损区进一步缩小，未见连续性骨痂，骨髓腔封闭，为骨不连。PLA组术后4w骨缺损两端有少量低密度的骨痂形成，缺损区呈现密度低于骨干的雾状影；术后8w，两断端在近尺骨侧出现高密度骨痂连接，远离尺骨侧骨缺损仍清晰可见；术后12w，骨缺损两端基本连接，骨密度不均，周围有骨痂形成，骨髓腔没有再通。PLA／BMG组术4周两断端有骨痂包裹，缺损区呈现大片云雾状高密度影，骨缺损区不易辨认；术后8w，骨缺损两端均基本连接，缺损区内呈现高密度影；术后12w，新骨桥接骨缺损，密度较均一，皮质骨连续，形成连接性骨质，骨缺损基本完全愈合，髓腔再通。自体骨组术后4w骨缺损区自体骨仍清晰可见，截骨端有少量骨痂形成；术后8w，植入自体骨段与宿主骨两端连接，连接处呈现高密度影，有较多的骨痂形成；术后12w，新骨桥接骨缺损，密度较8周时增高，但不均匀，周围有大量骨痂形成，髓腔再通。X线评分：术后4w、8w、12w，PLA／BMG复合材料组(4.25、7.25、9.50)与自体骨组(4.50、8.00、10.25)在修复骨缺损上无显著性差异，而PLA／BMG组和自体骨组修复骨缺损效果优于空白对照组(0.25、0.50、1.25)和PLA组(1.00、3.50、4.25)，存在显著性差异(P＜0.05)；PLA组仅在第8w显示出修复效果好于空白对照组(P＜0.05)，而4w与12w两组间无显著性差异。组织学观察：空白对照组骨缺损被纤维组织填充，无新骨组织形成。PLA组材料逐渐被纤维组织包裹吸收，成骨不活跃；PLA／BMG组材料可见大量新生骨向材料内部长入，间充质细胞向BMG贴附，材料与宿主骨结合紧密；自体骨组材料结合部有大量骨痂形成，骨组织已进行改建与重塑。
结论：PLA／BMG多孔复合材料与自体骨具有相同的修复节段性骨缺损能力，优于PLA材料的修复能力。
Part 1 Preparation of the Porous PLA/BMG Composite Biomaterials and its Physical and Chemical Characteristics Study
Objective: To develop a novel porous poly (lactic-acid) (PLA)/bone matrix gelatin (BMG) bioactive composite biomaterial with supercritical carbon dioxide fluid technique (SC-CO_2) and to choose the optimal PLA/BMG proportion by evaluating its physical and chemical characteristics and cellular compatibility.
Methods: The bone allografts procured from the qualified donor were processed into bone powder with the size of 50μm～200μm by deep-freezing, cleaning according to the processing standards of bone allograft in Shanxi Provincial Tissue Bank. Then the powder was processed into Bone Matrix Gelatin (BMG) by defatting and demineralization as described by Urist. The PLA and BMG, mixed with the PLA:BMG proportion of 9: 1、8: 2, 7: 3, 6: 4, and the 100μm～200μm NaCl was added in the SC-CO_2 equipment in order to make pores. Tightly closing the reactor kettle and CO_2 under 0℃was pumped into kettle. When the internal pressure reached 20MPa, the temperature was regulated at 36℃. The pressure was kept in (20±0.1) MPa and the temperature in (36±0.5)℃. After 30 minutes, the gas was released at the rate of 5 MPa/min.Opening the reacting kettle and the samples were taken out. After the samples were leached in deionised water to remove the NaCl in material, they were packaged and sterilized by radiation. The macroscopic observation, porosity, mechanics and scanning electron micrograph (SEM) were done to evaluate the physical and chemical properties. Then referring to the cellular compatibility, the optimal proportion was chosen.
Results: In macroscopic observation, the PLA/BMG composite biomaterial, prepared with the SC-CO_2 technique, have porous structure and good interconnective pores. The structure is like that of human cancellous bone. The porosity was more than 60 % and increasing with the BMG proportion being improved. While the compressive strength and elastic modulus was in inverse proportion to the proportion of BMG in PLA/BMG. When the BMG proportion was more than 30 %, the mechanics properties dropped significantly. In cell compatibility, PLA biomaterial did harm to proliferation of rat L6 cells. With the increasement of BMG proportion, cell compatibility was getting better. When the proportion reached 40 %, there was no difference between the PLA/BMG composite biomaterial group and the culture medium group. The SEM of PLA/BMG with 30 % BMG showed PLA and BMG were mixed uniformly, the pores diametesr were between 200μm and 400μm and the pores were distributed uniformly and interconnected each other. On the wall of pores, there still a number of about 10μm pores.
Conclusion: The percent of BMG in the BLA/BMG composite biomaterials prepared with SC-CO_2 technique was in direct proportion to its cellular compatibility and porosity, and in inverse proportion to its mechanics. The results show the PLA/BMG containing 30 % BMG was the optimal.
Part 2 Evaluation of Biocompatibility on the Porous PLA/BMG Composite Biomaterials
Objective: To evaluate the tissue biocompatibility in vivo by animal experiments and cell biocompatibility in vitro by cell experiments and provide the biocompatibility datas for PLA/BMG composite biomaterial.
Methods: Tests for cytotoxicity in vitro methods: Referring to the biological evaluation of medical devices-Tests for cytotoxicity: in vitro methods, the PLA/BMG and PLA porous biomaterials were immersed in DMEM culture medium with 0.1 g biomaterial each ml DMEM. After 72 hours in 37℃, the supernatants were collected by centrifuge. 200μl of the MC3T3-E1 cell suspension with a cell density of 5×10~5 cells/disk was seeded evenly into each well in 96-Well Plate. When the cell adhered after 24 hours, the culture medium in wells was removed and 200μl supernatant were added according to groups respectively, with DMEM culture medium being the control group and no cells well being the blank control group. The cells were cultured in 37℃humidified atmosphere of 5% CO_2. The cell culture mediums were exchanged every two days. On the 1~(st) day, 2~(nd) day, 3~(rd) day, 4~(th) day, 5~(th) day, 6~(th) day and 7~(th) day respectively, 30μl 2mg/ml MTT solution was added into each well. After being incubated for 4 hours, 150μl DMSO was added and the plate was shaken for 10 minutes. The OD of the medium was read at 570 nm with a plate reader. The cell proliferation rates were calculated and transformed into the toxicity grades. The cellular affinity of PLA/BMG composite biomaterial: The 3 mm×3 mm×3 mm PLA/BMG blocks processed were placed in 96-Well Plate. 500μl of 1×10~4/ml MC3T3-E1 cell suspension was seeded into each well and DMEM was added to immerse the PLA/BMG blocks. Cell morphology and adhering to materials was observed on the 1~(st), 3~(rd), 5~(th) day after seeding under phase contrast microscope and on the 5~(th) day under SEM. Biological evaluation in vivo: 20 Wistar mice, PLA biomaterials were implanted both in nape subcutaneous tissue and thigh muscle on the left side, and PLA/BMG biomaterials on the right side. The gross observation about animal situations was made. At the 2~(nd), 4~(th), 6~(th), 8~(th) week after implantation, the 5 mice were chosen to be killed randomly. The implanted material and surrounding tissue were procured and stained with HE. The histological changes were made observation under light microscope.
Results: Tests for cytotoxicity in vitro methods: In PLA/BMG group, the cell proliferation rates were over 100 % and the cell cytotoxic grades were Grade 0 from the 1~(st) day to the 7~(th) day. While in PLA group, the cell proliferation rates were less than 100 % and the cell cytotoxic grades were Grade I on the 2~(nd), 4~(th) and 7~(th) day. The cellular affinity of PLA/BMG composite biomaterial: on the 1~(st) day after seeding, there were few cells on PLA/BMG. On the 3~(rd) day, a few cells adhered to the edge of PLA/BMG and the cells surrounding kept normal morphology. On the 5~(th) day, the number of cell adhered to PLA/BMG increased, even converged somewhere. SEM showed there were cells both on the surface and in the pores of PLA/BMG. The cellular morphology was triangle or polygon with abundant microvillus on the surface. Some cells connected and overlapped by protrusions. The cells adhered to the surface of PLA/BMG tightly. Some cells were keeping at mitosis phase. Biological evaluation in vivo showed PLA were enclosed with connective tissue containing a lot of lymphocytes and neutrophilic granulocytes. The cells and tissue grew into PLA slowly. The PLA/BMG materials were enclosed with little connective tissue including a few inflammatory cells. The surrounding tissue immersed into the center of PLA/BMG easily. Some mesenchymal cells migrated to the edge of BMG.
Conclusion: the PLA/BMG composite biomaterials prepared with SC-CO_2 technique is of nice cellular affinity. The cytotoxicity grade is Grade 0, better than that of PLA. It shows good cell compatibility. Biological evaluation in vivo also suggests the tissue compatibility of PLA/BMG is better than that of PLA.
Part 3 Experimental Study on Osteoinductive Activity of the Porous PLA/BMG Composite Biomaterials
Objective: To evaluate the osteoinductive activity of PLA/BMG by calcium depositions, ALP activity and Ca content by Mouse osteoblast-like MC3T3-E1 cells be cultured in DMEM with the different biomaterials.
Methods: The PLA/BMG group: with 100μg of the scrushed PLA/BMG each well; The PLA group: with 100μg of the scrushed PLA each well. The DMEM group serves the control. There were 6 wells in each group. Cells suspensions were prepared and adjusted to 2×10~6 cells/ml. 20μl cells suspension was delivered into each well of 24 well plate with the different group materials. After the cells were adhered, 1 ml of culture medium containing 10μmol/ml disodiumβ-glycerophosphate (β-GP) and 5μg/ml vitamin C was added. 2/3 of Culture medium was changed every 3 days. After 2 weeks, the cells were collected. Calcification depositions assay: After being washed three times with PBS buffer, the cell cultures were fixed with formalin and stained by alizarin red solution. The microscopic images were taken with the converse phase microscope and the macroscopic ones with the digital camera. The percentage of the stained area, standing for the calcification deposition, was measured with the image processing and analysis software. ALP activity and Calcium content measurement: After the harvested cells were washed twice with Hanks solution. The cells were resuspended in 1 ml of 0.2 % Nonidet P-40 (NP-40) and sonicated in an ice bath for 2 min using an ultrasonic processor. The measurement of the ALP activity and calcium content were taken according to the instructions of kits.
Results: The ALP activity, Ca content and percent of calcification area in PLA/BMG group [ (325.59±70.40) U/g prot、(3.51±1.64) mmol/g prot)、(42.98±4.44) % ] were more than those in PLA group [ (63.62±30.01) U/gprot、(1.04±0.21) mmol/g prot, (9.55±1.94)% ] and DMEM control group [ (2.40±1.47) U/g prot、(0.70±0.24) mmol/g prot、(0.86±0.41) % ] (P＜0.05). Meanwhile, there was statistic difference between PLA group and DMEM group in the ALP activity and calcification area.
Conclusion: The PLA/BMG prepared by SC-CO_2 technique has good osteoinductive activity, better than that of PLA. And it is worth studying further as bone biomaterial and bone tissue engineering scaffold.
Part 4 Experimental Study of the Porous PLA/BMG Composite Biomaterials
for the Repair Radial Segmental Bone Defect of Rabbit
Objective: To evaluate the repair of rabbit radial defects by PLA/BMG biomaterials prepared by SC-CO_2 technique and provide the experimental date for the possible clinical applications.
Methods: Twenty four New Zealand male rabbits with the Wt of about 2.5 kg were obtained for this study. After the rabbits were anesthetized and the forelegs were sterilized, the diaphysis of both radii were exposed through a longitudinal extensile incision, the periosteum was elevated circumferentially, and the 12 mm long middle shaft of the radius was cut with a power-driven oscillating saw. Then a 12 mm bone defect was created. The biomaterials were filled according to the different groups. Then the wound was closed in layers and the animals were put back into their cages. The animals were divided into 2 groups randomly. In one group, nothing was filled in defect on the left and the bone autograft cut was re-implanted on the right; in another group, PLA/BMG was filled on the left and PLA/BMG was done on the right. 4 animals of each group were put to death at week 4, 8, 12 after operation. Radiographic observation and evaluation: Radiographs were taken when the animals were put to death at week 4, 8, 12 respectively. The situation of bone reconstruction were observed each group and evaluated in a blind condition according to the Standard of Lane's X-ray Scores. Histological observation: the radial samples from rabbits were harvested and fixed in formalin solution. Then the dehydrated samples were embedded with plastic and sectioned longitudinally by 5 mm thickness, stained with hematoxylin and eosin, and finally observed under light microscope for the formation of the new bone and the degradation of the material.
Results: Gross observation: except forl bone fracture in the blank control group and 2 fracture in the PLA group, all the animal were in good condition after operation and the operated limbs was uneventful with no detectable complications. Radiographic findings: In the blank control group, at 4 week postoperatively, little callus formed and the bone defect was clear; at 8 week postoperatively, a little callus with low-density was seen and the length of bone defect became shorter; at 12 week postoperatively, some slim callus formed at the both ends of broken bone and the defect was seen easily. In the PLA group, at 4 week postoperatively, a little callus with low-density formed at the ends and some fog-like image was seen in the defect; At 8 week postoperatively, the callus at the both ends met near to the ulna, but the defect was clear far to the ulna; at 12 week postoperatively, the both ends were connected with uneven shadow, little callus formed circumferentially and the cavitas medullaris did not recanalize. In the PLA/BMG group, at 4 week postoperatively, the both ends were enclosed with callus and the defect full of high-density shadow can not be identified easily; at 8 week postoperatively, the both ends met by high-density shadow; at 12 week postoperatively, with the new bone with uniform density bridging the both ends, the bone was reconstructed and the cavitas medullaris recanalized. In the autograft group, at 4 week postoperatively, the implanted bone could be identified and some callus formed at the broken ends; at 8 weeks postoperatively, the implanted bone were connected with the host bone by high-density callus; at 12 week postoperatively, the both ends were bridged with uneven high-density callus and the avitas medullaris recanalized. Radiographic evaluation: The PLA/BMG group and bone autograft group had no statistic difference at week 4, 8, 12 postoperatively. Which was better than both the PLA group and the blank control group (P＜0.05). There was significant difference at week 8 (P＜0.05) and no difference at week 4 and 8 between the PLA group and the blank control group. Histological Findings: In the blank group, the defect was filled with fiber connective tissue and no new bone formed. In the PLA group, the PLA were enveloped with connective tissue with little new bone formation. In the PLA/BMG group, the PLA/BMG incorporated with the host bone tightly. The new bone immersed into the PLA/BMG and some mesenchymal cells attached the BMG. In bone autograft group, large amounts of callus formed at the interface and the bone had reconstructed and remodeled.
Conclusions: The ability to repair bone defect of the PLA/BMG is same to that of bone autograft and better than that of the PLA.
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